Phytochrome is a photoreceptor for photomorphogenic responses in plants. That is, phytochrome detects the natural radiation of the plant environment and regulates adaptive growth and developmental responses critical for competition and survival. Phytochrome exists in two photointerconvertible forms: a red-light-absorbing form (Pr) and a far-red-light-absorbing form (Pfr). Thereby, the phytochrome system detects the ratio of red light (R) relative to far-red light (FR) and initiates appropriate morphological adaptation. For example, a low R:FR ratio could indicate the presence of shade from neighboring plants and initiate competitive morphological responses (e.g., increased shoot extension rate).
Historically, most of the understanding of phytochrome derived from the study of this chromoprotein as isolated from etiolated (i.e., dark-grown) plants, rather than from green (i.e., light-grown) plants. This is because phytochrome is relatively abundant in etiolated plants, while it is present in green plants in about 50-fold lower quantity. Further, the presence of chlorophyll in green plants makes the spectral assay of phytochrome impractical in green tissues.
Recent studies on phytochrome from green plants have indicated that it is different from phytochrome from etiolated plants. It has been hypothesized that green phytochrome and etiolated phytochrome could derive from different genes. Phytochrome of the type that is most abundant in etiolated plants is referred to herein by the term etiolated phytochrome, and, conversely, that which is most abundant in green plants is referred to herein by the term green phytochrome. However, the use of these terms is not meant to imply that there is no green phytochrome in etiolated plants, nor that there is no etiolated phytochrome in green plants. In fact, it has been observed in oat plants that etiolated plants contain a small amount of green phytochrome. (Shimazaki, Y. and L. H. Pratt, Planta 164 333-344, 1985).
Monoclonal antibodies directed to etiolated-oat phytochrome have been previously obtained and it has been demonstrated that most do not bind green-oat phytochrome (Tokuhisa, J. G., et al., Planta 164, 321-332, 1985; Shimazaki, Y. and L. H. Pratt, Planta 164 333-344, 1985). One monoclonal antibody directed to etiolated-pea phytochrome (Pea-25) is able to cross-react with at least some of the phytochrome from green oat shoots. The production of monoclonal antibodies specific for green phytochrome and that do not cross-react significantly with etiolated phytochrome has proven to be difficult because of the fact that green phytochrome is a very low abundance chromoprotein, estimated to be about 0.002% of extractable protein. Moreover, it is a very labile protein, and prior purification attempts yielded a relatively poor immunogen. Apparently, immunodominant contaminants were present in the prior method for green phytochrome purification and generated monoclonal antibodies directed primarily to the immunodominant contaminants.
Two hybridomas (designated GO-1 and GO-2) that were previously reported by the Applicants of the present invention (Pratt, L. H., and M.-M. Cordonnier, In Phytochrome and Photoregulation in Plants, Edited by M. Furuya, pp. 83-94, Academic Press, Tokyo, 1987) were prepared by immunizing with partially-purified green-phytochrome immunogen that was purified through hydroxyapatite chromatography as described by Shimazaki and Pratt (1985), and additionally purified by DEAE chromatography prior to electrophoretic purification. However, the GO-1 and GO-2 hybridoma cell lines have proven to be not sufficiently specific for green phytochrome to serve as the needed probes for resolution of phytochrome into individual species and identification of phytochrome proteins encoded by separate phytochrome genes. GO-1 by direct Elisa tested against etiolated phytochrome vs green phytochrome reacted better with etiolated phytochrome than green phytochrome. The Elisa data for GO-2 indicated that its activity was no more specific than a non-immune mouse control.